Pulmonary vascular disease is perhaps the most important complication for children with common congenital heart defects (CHD) that result in increased pulmonary blood flow (PBF) and pressure, such as large ventricular septal defects (VSD) and atrioventricular septal defects (AVSD). Beginning immediately after birth, the pulmonary vasculature in these patients is subjected to pathologic mechanical forces, such as increased shear stress, resulting in early functional abnormalities of the vascular endothelium, including decreased bioavailable nitric oxide (NO). Repairing these defects in infancy or early childhood mostly prevents the development of irreversible pulmonary vascular disease, however perioperative pulmonary vascular dysfunction, particularly following cardiopulmonary bypass (CPB), continues to contribute to the morbidity and mortality of these patients. Utilizing our clinically relevant lamb model of CHD, we have generated compelling data demonstrating that increased PBF impairs carnitine homeostasis, in association with mitochondrial dysfunction, decreased HSP90/eNOS interactions, increased reactive oxygen species (ROS) production, and decreased bioavailable NO, and that eNOS mitochondrial translocation and the loss of CrAT activity is pivotal to this pathobiology. In addition, our preliminary data indicate that treatment with exogenous L-carnitine can attenuate these effects. Importantly, we also have novel preliminary data demonstrating altered carnitine homeostasis, mitochondrial dysfunction, and ROS production in infants with VSDs. Thus, we hypothesize that: (1) increased PBF associated with CHD induces early aberrations in carnitine homeostasis that ultimately result in decreased bioavailable NO and altered pulmonary vascular function; and (2) L-carnitine treatment can attenuate this pathobiology and improve clinical outcomes. In order to test these hypotheses, we propose a translational project that integrates cell culture, animal and human based studies to accomplish the following aims: (1) To determine the effect of L-carnitine treatment on carnitine homeostasis, mitochondrial function, ROS production, bioavailable NO, and vascular reactivity in a lamb model of CHD; (2) To determine the effect of acute IV L-carnitine treatment on pulmonary vascular resistance, vascular reactivity, and myocardial performance following CPB in a lamb model of CHD; (3A) To determine perioperative alterations in carnitine homeostasis, mitochondrial function, ROS production, and NO bioavailability in infants with VSD and AVSD; and (3B) To pilot a trial assessing the safety and pharmacokinetics of perioperative IV L-carnitine administration in these patients, and begin to assess its effect on post-operative clinical outcomes. Importantly, L-carnitine has been administered to children for decades, with an excellent safety profile, and can be delivered orally as well as IV. Taken together, our pre-clinical and preliminary human data and the feasibility of L-carnitine therapy represent an important opportunity to translate basic mechanisms into a novel targeted therapeutic strategy for pulmonary vascular disease in a high-risk patient population.